Receptor-mediated chitin perception in legume roots is functionally separable from Nod factor perception
Edited by Richard A. Dixon, University of North Texas, Denton, TX, and approved August 17, 2017 (received for review April 26, 2017)
Significance
Like 80–90% of land plants, legumes form endosymbioses with arbuscular mycorrhizal fungi, host endophytes, support a rhizosphere community, and are attacked by pathogens. The ability of root cells to distinguish between these soil microbes and the mixture of chitinaceous compounds they display as signal molecules is important for an appropriate plant response. We show that legumes possess very similar receptors enabling root cells to separate perception of chitin, which triggers responses to pathogens, from perception of lipochitin oligosaccharides (Nod factors), which trigger endosymbiosis with rhizobial bacteria. The chitin receptors bind chitin in biochemical assays, and inactivation of the corresponding genes impairs defense responses toward pathogens. Together this establishes a long-sought foundation for dissecting plants’ response mechanisms toward different soil microbes.
Abstract
The ability of root cells to distinguish mutualistic microbes from pathogens is crucial for plants that allow symbiotic microorganisms to infect and colonize their internal root tissues. Here we show that Lotus japonicus and Medicago truncatula possess very similar LysM pattern-recognition receptors, LjLYS6/MtLYK9 and MtLYR4, enabling root cells to separate the perception of chitin oligomeric microbe-associated molecular patterns from the perception of lipochitin oligosaccharide by the LjNFR1/MtLYK3 and LjNFR5/MtNFP receptors triggering symbiosis. Inactivation of chitin-receptor genes in Ljlys6, Mtlyk9, and Mtlyr4 mutants eliminates early reactive oxygen species responses and induction of defense-response genes in roots. Ljlys6, Mtlyk9, and Mtlyr4 mutants were also more susceptible to fungal and bacterial pathogens, while infection and colonization by rhizobia and arbuscular mycorrhizal fungi was maintained. Biochemical binding studies with purified LjLYS6 ectodomains further showed that at least six GlcNAc moieties (CO6) are required for optimal binding efficiency. The 2.3-Å crystal structure of the LjLYS6 ectodomain reveals three LysM βααβ motifs similar to other LysM proteins and a conserved chitin-binding site. These results show that distinct receptor sets in legume roots respond to chitin and lipochitin oligosaccharides found in the heterogeneous mixture of chitinaceous compounds originating from soil microbes. This establishes a foundation for genetic and biochemical dissection of the perception and the downstream responses separating defense from symbiosis in the roots of the 80–90% of land plants able to develop rhizobial and/or mycorrhizal endosymbiosis.
Data Availability
Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.wwpdb.org (PDB ID code 5LS2).
Acknowledgments
We thank Finn Pedersen and Karina Kristensen for taking care of plants in the greenhouse. This work was supported by Danish National Research Foundation Grant DNRF79, by the Bill and Melinda Gates Foundation as part of Engineering the Nitrogen Symbiosis for Africa, and by Biotechnology and Biological Sciences Research Council Grant BB/J004553/1.
Supporting Information
Appendix (PDF)
- Download
- 6.78 MB
References
1
GED Oldroyd, JD Murray, PS Poole, JA Downie, The rules of engagement in the legume-rhizobial symbiosis. Annu Rev Genet 45, 119–144 (2011).
2
M Parniske, Arbuscular mycorrhiza: The mother of plant root endosymbioses. Nat Rev Microbiol 6, 763–775 (2008).
3
CD Bordenave, et al., Defense responses in two ecotypes of Lotus japonicus against non-pathogenic Pseudomonas syringae. PLoS One 8, e83199 (2013).
4
C Ben, et al., Natural diversity in the model legume Medicago truncatula allows identifying distinct genetic mechanisms conferring partial resistance to Verticillium wilt. J Exp Bot 64, 317–332 (2013).
5
C Ben, et al., MtQRRS1, an R-locus required for Medicago truncatula quantitative resistance to Ralstonia solanacearum. New Phytol 199, 758–772 (2013).
6
R Zgadzaj, et al., A legume genetic framework controls infection of nodules by symbiotic and endophytic bacteria. PLoS Genet 11, e1005280 (2015).
7
R Zgadzaj, et al., Root nodule symbiosis in Lotus japonicus drives the establishment of distinctive rhizosphere, root, and nodule bacterial communities. Proc Natl Acad Sci USA 113, E7996–E8005 (2016).
8
MC Peck, RF Fisher, SR Long, Diverse flavonoids stimulate NodD1 binding to nod gene promoters in Sinorhizobium meliloti. J Bacteriol 188, 5417–5427 (2006).
9
JE Cooper, Early interactions between legumes and rhizobia: Disclosing complexity in a molecular dialogue. J Appl Microbiol 103, 1355–1365 (2007).
10
J Dénarié, F Debellé, J-C Promé, Rhizobium lipo-chitooligosaccharide nodulation factors: Signaling molecules mediating recognition and morphogenesis. Annu Rev Biochem 65, 503–535 (1996).
11
R Catoira, et al., The HCL gene of Medicago truncatula controls Rhizobium-induced root hair curling. Development 128, 1507–1518 (2001).
12
EB Madsen, et al., A receptor kinase gene of the LysM type is involved in legume perception of rhizobial signals. Nature 425, 637–640 (2003).
13
E Limpens, et al., LysM domain receptor kinases regulating rhizobial Nod factor-induced infection. Science 302, 630–633 (2003).
14
S Radutoiu, et al., Plant recognition of symbiotic bacteria requires two LysM receptor-like kinases. Nature 425, 585–592 (2003).
15
BB Amor, et al., The NFP locus of Medicago truncatula controls an early step of Nod factor signal transduction upstream of a rapid calcium flux and root hair deformation. Plant J 34, 495–506 (2003).
16
J-F Arrighi, et al., The Medicago truncatula lysin [corrected] motif-receptor-like kinase gene family includes NFP and new nodule-expressed genes. Plant Physiol 142, 265–279 (2006).
17
LH Madsen, et al., The molecular network governing nodule organogenesis and infection in the model legume Lotus japonicus. Nat Commun 1, 10 (2010).
18
CH Haney, et al., Symbiotic rhizobia bacteria trigger a change in localization and dynamics of the Medicago truncatula receptor kinase LYK3. Plant Cell 23, 2774–2787 (2011).
19
EB Madsen, et al., Autophosphorylation is essential for the in vivo function of the Lotus japonicus Nod factor receptor 1 and receptor-mediated signalling in cooperation with Nod factor receptor 5. Plant J 65, 404–417 (2011).
20
A Broghammer, et al., Legume receptors perceive the rhizobial lipochitin oligosaccharide signal molecules by direct binding. Proc Natl Acad Sci USA 109, 13859–13864 (2012).
21
DW Ehrhardt, R Wais, SR Long, Calcium spiking in plant root hairs responding to Rhizobium nodulation signals. Cell 85, 673–681 (1996).
22
GED Oldroyd, JA Downie, Nuclear calcium changes at the core of symbiosis signalling. Curr Opin Plant Biol 9, 351–357 (2006).
23
H Miwa, J Sun, GED Oldroyd, JA Downie, Analysis of Nod-factor-induced calcium signaling in root hairs of symbiotically defective mutants of Lotus japonicus. Mol Plant Microbe Interact 19, 914–923 (2006).
24
SJ Kelly, et al., Conditional requirement for exopolysaccharide in the Mesorhizobium-Lotus symbiosis. Mol Plant Microbe Interact 26, 319–329 (2013).
25
Y Kawaharada, et al., Receptor-mediated exopolysaccharide perception controls bacterial infection. Nature 523, 308–312 (2015).
26
Y Kawaharada, et al., Differential regulation of the Epr3 receptor coordinates membrane-restricted rhizobial colonization of root nodule primordia. Nat Commun 8, 14534 (2017).
27
CHSG Meneses, LFM Rouws, JL Simoes-Araujo, MS Vidal, JI Baldani, Exopolysaccharide production is required for biofilm formation and plant colonization by the nitrogen-fixing endophyte Gluconacetobacter diazotrophicus. Mol Plant Microbe Interact 24, 1448–1458 (2011).
28
M Buee, M Rossignol, A Jauneau, R Ranjeva, G Bécard, The pre-symbiotic growth of arbuscular mycorrhizal fungi is induced by a branching factor partially purified from plant root exudates. Mol Plant Microbe Interact 13, 693–698 (2000).
29
A Genre, M Chabaud, T Timmers, P Bonfante, DG Barker, Arbuscular mycorrhizal fungi elicit a novel intracellular apparatus in Medicago truncatula root epidermal cells before infection. Plant Cell 17, 3489–3499 (2005).
30
K Akiyama, K Matsuzaki, H Hayashi, Plant sesquiterpenes induce hyphal branching in arbuscular mycorrhizal fungi. Nature 435, 824–827 (2005).
31
F Maillet, et al., Fungal lipochitooligosaccharide symbiotic signals in arbuscular mycorrhiza. Nature 469, 58–63 (2011).
32
A Genre, et al., Short-chain chitin oligomers from arbuscular mycorrhizal fungi trigger nuclear Ca2+ spiking in Medicago truncatula roots and their production is enhanced by strigolactone. New Phytol 198, 190–202 (2013).
33
R Op den Camp, et al., LysM-type mycorrhizal receptor recruited for rhizobium symbiosis in nonlegume Parasponia. Science 331, 909–912 (2011).
34
L Buendia, T Wang, A Girardin, B Lefebvre, The LysM receptor-like kinase SlLYK10 regulates the arbuscular mycorrhizal symbiosis in tomato. New Phytol 210, 184–195 (2016).
35
JDG Jones, JL Dangl, The plant immune system. Nature 444, 323–329 (2006).
36
H Kaku, et al., Plant cells recognize chitin fragments for defense signaling through a plasma membrane receptor. Proc Natl Acad Sci USA 103, 11086–11091 (2006).
37
A Miya, et al., CERK1, a LysM receptor kinase, is essential for chitin elicitor signaling in Arabidopsis. Proc Natl Acad Sci USA 104, 19613–19618 (2007).
38
J Wan, et al., A LysM receptor-like kinase plays a critical role in chitin signaling and fungal resistance in Arabidopsis. Plant Cell 20, 471–481 (2008).
39
T Shimizu, et al., Two LysM receptor molecules, CEBiP and OsCERK1, cooperatively regulate chitin elicitor signaling in rice. Plant J 64, 204–214 (2010).
40
Y Cao, et al., The kinase LYK5 is a major chitin receptor in Arabidopsis and forms a chitin-induced complex with related kinase CERK1. Elife 3, e03766 (2014).
41
G Carotenuto, et al., The rice LysM receptor-like kinase OsCERK1 is required for the perception of short-chain chitin oligomers in arbuscular mycorrhizal signaling. New Phytol 214, 1440–1446 (2017).
42
R Willmann, et al., Arabidopsis lysin-motif proteins LYM1 LYM3 CERK1 mediate bacterial peptidoglycan sensing and immunity to bacterial infection. Proc Natl Acad Sci USA 108, 19824–19829 (2011).
43
K Miyata, et al., The bifunctional plant receptor, OsCERK1, regulates both chitin-triggered immunity and arbuscular mycorrhizal symbiosis in rice. Plant Cell Physiol 55, 1864–1872 (2014).
44
X Zhang, et al., The receptor kinase CERK1 has dual functions in symbiosis and immunity signalling. Plant J 81, 258–267 (2015).
45
Y Liang, et al., Nonlegumes respond to rhizobial Nod factors by suppressing the innate immune response. Science 341, 1384–1387 (2013).
46
X-C Zhang, et al., Molecular evolution of lysin motif-type receptor-like kinases in plants. Plant Physiol 144, 623–636 (2007).
47
S-H Shiu, et al., Comparative analysis of the receptor-like kinase family in Arabidopsis and rice. Plant Cell 16, 1220–1234 (2004).
48
GV Lohmann, et al., Evolution and regulation of the Lotus japonicus LysM receptor gene family. Mol Plant Microbe Interact 23, 510–521 (2010).
49
X-C Zhang, SB Cannon, G Stacey, Evolutionary genomics of LysM genes in land plants. BMC Evol Biol 9, 183 (2009).
50
DF Urbański, A Małolepszy, J Stougaard, SU Andersen, Genome-wide LORE1 retrotransposon mutagenesis and high-throughput insertion detection in Lotus japonicus. Plant J 69, 731–741 (2012).
51
E Fukai, et al., Establishment of a Lotus japonicus gene tagging population using the exon-targeting endogenous retrotransposon LORE1. Plant J 69, 720–730 (2012).
52
M Tadege, et al., Large-scale insertional mutagenesis using the Tnt1 retrotransposon in the model legume Medicago truncatula. Plant J 54, 335–347 (2008).
53
A Małolepszy, et al., The LORE1 insertion mutant resource. Plant J 88, 306–317 (2016).
54
CJ Wienken, P Baaske, U Rothbauer, D Braun, S Duhr, Protein-binding assays in biological liquids using microscale thermophoresis. Nat Commun 1, 100 (2010).
55
T Liu, et al., Chitin-induced dimerization activates a plant immune receptor. Science 336, 1160–1164 (2012).
56
JEMM Wong, et al., An intermolecular binding mechanism involving multiple LysM domains mediates carbohydrate recognition by an endopeptidase. Acta Crystallogr D Biol Crystallogr 71, 592–605 (2015).
57
A Sánchez-Vallet, et al., Fungal effector Ecp6 outcompetes host immune receptor for chitin binding through intrachain LysM dimerization. Elife 2, e00790 (2013).
58
S Sato, et al., Genome structure of the legume, Lotus japonicus. DNA Res 15, 227–239 (2008).
59
H Tang, et al., An improved genome release (version Mt4.0) for the model legume Medicago truncatula. BMC Genomics 15, 312 (2014).
60
H Yoshioka, et al., Induction of plant gp91 phox homolog by fungal cell wall, arachidonic acid, and salicylic acid in potato. Mol Plant Microbe Interact 14, 725–736 (2001).
61
J Li, G Brader, ET Palva, The WRKY70 transcription factor: A node of convergence for jasmonate-mediated and salicylate-mediated signals in plant defense. Plant Cell 16, 319–331 (2004).
62
X Shi, Z Tian, J Liu, EAG van der Vossen, C Xie, A potato pathogenesis-related protein gene, StPRp27, contributes to race-nonspecific resistance against Phytophthora infestans. Mol Biol Rep 39, 1909–1916 (2012).
63
N Zhou, TL Tootle, F Tsui, DF Klessig, J Glazebrook, PAD4 functions upstream from salicylic acid to control defense responses in Arabidopsis. Plant Cell 10, 1021–1030 (1998).
64
J Wan, S Zhang, G Stacey, Activation of a mitogen-activated protein kinase pathway in Arabidopsis by chitin. Mol Plant Pathol 5, 125–135 (2004).
65
E Limpens, et al., Cell- and tissue-specific transcriptome analyses of Medicago truncatula root nodules. PLoS One 8, e64377 (2013).
66
RA Cabeza, et al., RNA-seq transcriptome profiling reveals that Medicago truncatula nodules acclimate N2 fixation before emerging P deficiency reaches the nodules. J Exp Bot 65, 6035–6048 (2014).
67
F Berrabah, P Ratet, B Gourion, Multiple steps control immunity during the intracellular accommodation of rhizobia. J Exp Bot 66, 1977–1985 (2015).
68
M Giovannetti, A Mari, M Novero, P Bonfante, Early Lotus japonicus root transcriptomic responses to symbiotic and pathogenic fungal exudates. Front Plant Sci 6, 480 (2015).
69
E Iizasa, M Mitsutomi, Y Nagano, Direct binding of a plant LysM receptor-like kinase, LysM RLK1/CERK1, to chitin in vitro. J Biol Chem 285, 2996–3004 (2010).
70
CT Pedersen, et al., N-glycan maturation mutants in Lotus japonicus for basic and applied glycoprotein research. Plant J 91, 394–407 (2017).
71
P Bueno, et al., Time-course of lipoxygenase, antioxidant enzyme activities and H2O2 accumulation during the early stages of Rhizobium–legume symbiosis. New Phytol 152, 91–96 (2001).
72
R Santos, D Hérouart, S Sigaud, D Touati, A Puppo, Oxidative burst in alfalfa-Sinorhizobium meliloti symbiotic interaction. Mol Plant Microbe Interact 14, 86–89 (2001).
73
SK Ramu, H-M Peng, DR Cook, Nod factor induction of reactive oxygen species production is correlated with expression of the early nodulin gene rip1 in Medicago truncatula. Mol Plant Microbe Interact 15, 522–528 (2002).
74
S Peleg-Grossman, H Volpin, A Levine, Root hair curling and Rhizobium infection in Medicago truncatula are mediated by phosphatidylinositide-regulated endocytosis and reactive oxygen species. J Exp Bot 58, 1637–1649 (2007).
75
L Cárdenas, A Martínez, F Sánchez, C Quinto, Fast, transient and specific intracellular ROS changes in living root hair cells responding to Nod factors (NFs). Plant J 56, 802–813 (2008).
76
T Nakagawa, et al., From defense to symbiosis: Limited alterations in the kinase domain of LysM receptor-like kinases are crucial for evolution of legume-Rhizobium symbiosis. Plant J 65, 169–180 (2011).
77
S De Mita, A Streng, T Bisseling, R Geurts, Evolution of a symbiotic receptor through gene duplications in the legume-Rhizobium mutualism. New Phytol 201, 961–972 (2014).
78
DWA Buchan, F Minneci, TCO Nugent, K Bryson, DT Jones, Scalable web services for the PSIPRED protein analysis workbench. Nucleic Acids Res 41, W349–W357 (2013).
79
TN Petersen, S Brunak, G von Heijne, H Nielsen, SignalP 4.0: Discriminating signal peptides from transmembrane regions. Nat Methods 8, 785–786 (2011).
80
PD Adams, et al., The Phenix software for automated determination of macromolecular structures. Methods 55, 94–106 (2011).
81
P Emsley, B Lohkamp, WG Scott, K Cowtan, Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66, 486–501 (2010).
82
X Robert, P Gouet, Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res 42, W320–W324 (2014).
83
LA Kelley, S Mezulis, CM Yates, MN Wass, MJE Sternberg, The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 10, 845–858 (2015).
84
T Mun, A Bachmann, V Gupta, J Stougaard, SU Andersen, Lotus base: An integrated information portal for the model legume Lotus japonicus. Sci Rep 6, 39447 (2016).
85
K Handberg, J Stougaard, Lotus japonicus, an autogamous, diploid legume species for classical and molecular genetics. Plant J 2, 487–496 (1992).
86
H Vierheilig, AP Coughlan, U Wyss, Y Piché, Ink and vinegar, a simple staining technique for arbuscular-mycorrhizal fungi. Appl Environ Microbiol 64, 5004–5007 (1998).
87
A Trouvelot, JL Kough, V Gianinazzi-Pearson, Mesure du taux de mycorhization VA d’un systeme radiculaire. Recherche de methods d’estimation ayant une signification fonctionnelle. Physiological and Genetical Aspects of Mycorrhizae, eds V Gianinazzi-Pearson, S Gianinazzi (INRA, Paris), pp. 217–221 (1986).
88
C Engler, R Kandzia, S Marillonnet, A one pot, one step, precision cloning method with high throughput capability. PLoS One 3, e3647 (2008).
89
J Stougaard, D Abildsten, KA Marcker, The Agrobacterium rhizogenes pRi TL-DNA segment as a gene vector system for transformation of plants. Mol Gen Genet 207, 251–255 (1987).
90
J Hansen, JE Jørgensen, J Stougaard, KA Marcker, Hairy roots–A short cut to transgenic root nodules. Plant Cell Rep 8, 12–15 (1989).
91
K Tamura, G Stecher, D Peterson, A Filipski, S Kumar, MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30, 2725–2729 (2013).
92
SR Lloyd, H-J Schoonbeek, M Trick, C Zipfel, CJ Ridout, Methods to study PAMP-triggered immunity in Brassica species. Mol Plant Microbe Interact 27, 286–295 (2014).
93
C Gachon, P Saindrenan, Real-time PCR monitoring of fungal development in Arabidopsis thaliana infected by Alternaria brassicicola and Botrytis cinerea. Plant Physiol Biochem 42, 367–371 (2004).
Information & Authors
Information
Published in
Classifications
Data Availability
Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.wwpdb.org (PDB ID code 5LS2).
Submission history
Published online: September 5, 2017
Published in issue: September 19, 2017
Keywords
Acknowledgments
We thank Finn Pedersen and Karina Kristensen for taking care of plants in the greenhouse. This work was supported by Danish National Research Foundation Grant DNRF79, by the Bill and Melinda Gates Foundation as part of Engineering the Nitrogen Symbiosis for Africa, and by Biotechnology and Biological Sciences Research Council Grant BB/J004553/1.
Notes
This article is a PNAS Direct Submission.
Authors
Competing Interests
The authors declare no conflict of interest.
Metrics & Citations
Metrics
Citation statements
Altmetrics
Citations
Cite this article
114 (38) E8118-E8127,
Export the article citation data by selecting a format from the list below and clicking Export.
Cited by
Loading...
View Options
View options
PDF format
Download this article as a PDF file
DOWNLOAD PDFLogin options
Check if you have access through your login credentials or your institution to get full access on this article.
Personal login Institutional LoginRecommend to a librarian
Recommend PNAS to a LibrarianPurchase options
Purchase this article to access the full text.